Abstract 5393: C5a complement enhances generation of APC-induced Foxp3+ T regulatory cells

Abstract

Abstract Introduction: The complement system has complex interactions with the cellular immune system. Preliminary studies demonstrated that presence of complement significantly enhances the ability of malignant B cells and professional antigen-presenting cells to induce T regulatory (Treg) cells in vitro. The current study was designed to identify the specific complement component involved and to evaluate if complement depletion improves active immune responses through inhibition of Treg activity. Methods: Enriched CD4+ T cells from healthy human donors were co-cultured in vitro with primary myeloid dendritic cells (mDCs) in the presence of purified active complement proteins or unmodified human serum. The effect of serum treated with agents that block C5a generation, such as soluble complement receptor 1 (sCR1) that inhibits C5 convertase formation, or cobra venom factor (CVF) that enzymatically depletes C3 and C5, was also evaluated. Following culture, the percent of CD4+ T cells expressing Treg phenotype (CD3+CD4+CD25highFoxp3+) was evaluated by flow cytometry. In vivo, the effect of C5a inhibition on active immunization was also assessed following immunization of C57Bl/6 mice with a single, subcutaneous injection of ovalbumin (100 μg/mice) with or without agents that block C5a generation. Development of anti-ovalbumin antibody and anti-ovalbumin T cell responses were analyzed by ELISA and flow cytometry. Results: In vitro (human system): 1. In the presence of purified C5a protein, primary mDCs induced significantly more Tregs in co-cultured CD4+ T cells. Fewer Tregs were seen in the absence of complement, or with purified C1q, C3a, C3b or C5 proteins [p0.05 for Media vs other proteins]. 2. Serum C5a depletion or inhibition of C5a generation by anti-human C5a or sCR1 treatments decreased serum's ability to enhance mDC-induced Tregs [Intact serum vs depleted serum, p<0.05]. In vivo (murine system): 1. Circulating CD4+ T cells with T regulatory phenotype (Foxp3+) were significantly lower in mice injected with Ova plus CVF or sCR1 than mice injected with Ova alone [p=0.021 (CVF), 0.016 (sCR1)]. 2. Inhibition of C5a generation by sCR1 or CVF at the site of ovalbumin immunization resulted in higher anti-ovalbumin serum IgM levels [Ova alone vs Ova plus CVF or sCR1 – p=0.047 or p-0.014, respectively] and higher ovalbumin-specific CD8+ T cells in draining lymph nodes [Ova alone vs Ova plus CVF or sCR1 - p=0.048 or 0.041, respectively]. Conclusion: C5a complement plays a key role in the induction of Tregs induced by professional APCs. Inhibition of C5a generation at the site of immunization results in increased antigen-specific active immune responses. Ongoing studies are exploring the mechanisms responsible for the effect of C5a on APC-induced Treg generation. The data from this study has potential clinical applications, such as enhancing active immune responses against a broad variety of less immunogenic antigens, including cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5393. doi:1538-7445.AM2012-5393